This application is submitted in response to NIH Notice Number NOT-OD-09-058 entitled 'NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications'. Current techniques for full karyotype analysis rely on metaphase spreads, and cells in interphase cannot be analyzed. Since cells obtained from tumors are rarely in metaphase, our ongoing research focuses on the development of technologies for the cytogenetic analysis of interphase cells. The parent grant entitled 'Accelerating cancer research with single cell arrays (SCAs)'addresses the sensitive detection of chromosomal changes such as small translocations or genomic imbalances in small numbers of cells isolated from breast or thyroid cancer cell lines. The parent project will demonstrate the feasibility that interphase cell nuclei can be extended reproducibly on a solid support and develop methods for the cytogenetic analysis of extended chromatin fibers. This Competing Revision Application will define the working limits of the SCA technology by analyzing a range of relevant samples that has been processed under conditions that are likely to produce partial DNA degradation. On the other hand, our preliminary studies have demonstrated that the analysis of specimens which have undergone minimal degradation is complicated by hybridization of probes to DNA transcripts, i.e., RNA present in these cells. Thus, a second objective of the proposed research is the development of techniques to increase signal-to-noise ratios through RNA digestion or blocking. The two Specific Aims of the present application are 1. Define the limits of the SCA technology by analyzing samples that have undergone DNA degradation. Most archival samples will have suffered from fixation and storage. To investigate how widely the SCA technology can be applied to samples of interest to the cancer research community, we will study the effects of DNA nicks or loss and protein cross-links in archival samples comprised of acetic acid: methanol fixed lymphocytes, frozen placental tissues as well formalin-fixed paraffin-embedded tissue blocks. 2. Increase signal-to-noise ratios and thus the detection sensitivity by removal of RNA from chromatin. Using extended chromatin preparations from breast and thyroid cancer cells harvested from mammalian cell cultures, we will develop protocols to enzymatically digest and remove RNAs that interfere with the assay or to block hybridization of DNA probes to residual RNA, while minimizing DNA loss. SCAs will become powerful tools in basic and applied/clinical research, where such a sensitive assay may support cell classifications, thereby benefiting patients with de novo translocations or premalignant lesions as well as cancer patients. Furthermore, SCAs will allow the analysis of very small samples regardless of their viability or cell cycle stage. This will open new avenues for the analysis of small samples like those obtained by fine needle biopsies as well as the analysis of circulating or exfoliated tumor cells.